The present invention relates to an apparatus and to a process for continuous, cell-free, in vitro synthesis of proteins and polypeptides, particularly peptide-MHC complexes.
In vivo expression of foreign or synthetic genes, and overexpression of native genes by cells, are subject to a number of limitations. Many gene products are insoluble or unstable and either are de:graded by intracellular proteases or aggregate in inclusion bodies. Other products are toxic to the cell and cannot be expressed at all. Several regulatory proteins are unstable, and gene regulation mechanisms can also lead to poor levels of expression.
In vitro expression of proteins in a cell-free system avoids these and other problems. The absence of cellular control mechanisms and the ability to manipulate incubation mixture composition are advantages of this type of system. But low polypeptide yield has been a major obstacle with these systems. Early attempts to express polypeptides in cell-free systems typically produced only two to three polypeptide chains per mRNA chain used.
More recently, Spirin et al. have described a continuous cell-free translation system for producing polypeptides in higher yields. Science 242:1162-1163 (1988); see also Ryabova et al., Nucleic Acids Research 17:4412 (1989), and Baranov et al., Gene 84:463-466 (1989). The Spirin system uses an Amicon 8 MC micro-ultrafiltration device as a bioreactor for polypeptide synthesis. The device is a cylindrical flow cell with a 25 mm diameter membrane mounted at the bottom of the cell. The surface area of this membrane that is available for filtration is about 4 cm.sup.2 somewhat less than the 5 cm.sup.2 calculated based on its 25 mm diameter because of the O-ring used to mount the membrane in the device. A stirring bar is used to mix feed buffer and lysate contained in the cell.
Spirin's group has reported translation rates for globin of as much as 2 mg from 0.5 ml of rabbit reticulocyte lysate after 100 hours. See Ryabova et al., loc. cit. The volume of this liquid in the flow cell varies during operation, however, between about 1 and 5 ml, due to the compressibility of air in the chamber. The maximum flow rate possible is about 3 ml/hr and this flow rate cannot be maintained during continued operation, but tapers to 2 or even 1 ml/hr over the ensuing 4 to 5 hours. Thus, neither flow rate nor flow cell volume can be maintained during operation.
In addition to difficulties associated with controlling the flow rate and the volume of liquid contained in the flow cell, it also is impossible to control temperature and outgassing in this flow cell. Single parameters therefore cannot be varied systematically in this system. This means that optimization of a process for synthesis of a particular polypeptide is virtually impossible with this type of system.
A purported improvement of the Spirin system is described by Kigawa et al., in which the Amicon 8MC micro-ultrafiltration units was replaced with a reaction chamber with a capacity equal to the reaction mixture volume and in which a high performance liquid chromatography pump was used to supply the substrate solution. J. Biochem. 110:166-168 (1991). This system provided laboratory-scale polypeptide production, with only 0.1 mg of chloramphenicol acetyltransferase being synthesized in 17 hours from 1 ml of reaction mixture.